shRNA抑制c-fos表达对P-gp介导的乳腺癌多药耐药的影响

多药耐药(multidrug resistance,MDR)是导致化疗失败的重要原因,多药耐药基因(multidrug resistance gene,mdr1)产物P-糖蛋白(P-glycoprotein,P-gp)过表达是最主要的耐药机制。原癌基因c-fos在肿瘤MDR中的作用渐受重视。主要选用人乳腺癌敏感株MCF-7和阿霉素(adriamycin,ADR)筛选的、mdr1/P-gp高表达的耐药株MCF-7/ADR,探讨c-fos在P-gp介导的乳腺癌MDR中的作用。相对于MCF-7,c-fos在MCF-7/ADR高表达。采用shRNA法下调c-fos表达后,MCF-7/ADR对ADR的敏感性大大增强,且mdr1/P-gp表达减少、P-gp外排功能降低。c-fos表达下调可逆转对P-gp介导的乳腺癌MDR的实验结果,为c-fos成为逆转肿瘤耐药诊断和治疗的新靶标,对实现耐药乳腺癌的分子靶向治疗提供了理论基础。     

P-gp 和MDR1 在三种乳腺癌细胞中表达差异研究

目的:比较P-gp和MDR1在人乳腺癌敏感细胞(MCF-7/S)和耐药细胞(MCF-7/ADR、MCF-7/TAM)中的表达差异,初步探讨乳腺癌细胞对阿霉素与对三苯氧胺产生耐药机制的区别。方法:采用免疫细胞化学法、流式细胞术检测P-gp,采用实时荧光定量PCR法检测MDR1在三种乳腺癌细胞中的表达情况。结果:在MCF-7/ADR细胞中P-gp和MDR1均呈高表达,阳性表达率与MCF-7/S细胞比较,有统计学意义(P<0.01)。在MCF-7/TAM细胞中P-gp、MDR1均呈低表达,与MCF-7/S细胞比较,无统计学意义(P>0.05)。结论:P-gp和MDR1的高表达是乳腺癌细胞对阿霉素产生耐药的主要机制,而并非是乳腺癌细胞对三苯氧胺产生耐药的机制。     

PI3K/AKT 通路参与调控乳腺癌多药耐药和侵袭转移的研究

目的:探讨磷脂酰肌醇-3-激酶/丝苏氨酸蛋白激酶(phosphatidylinositol 3 kinase/serine-threonine kinase,PI3K/AKT)信号通路与乳腺癌多药耐药和侵袭转移的相关性。方法:以乳腺癌细胞系MCF-7为母本,持续低浓度加药诱导建立阿霉素(Adriamycin,ADR)耐药系MCF-7/ADR’。细胞免疫荧光检测两细胞系中磷酸化AKT(phosphorylated AKT,P-AKT)、P-糖蛋白(P-Glycoprotein,P-gp)、基质金属蛋白酶2(matrix metalloproteinase-2,MMP-2)的表达。PI3K抑制剂LY294002作用两系前后,Western Blot检测P-AKT、MMP-2、P-gp的表达改变及qRT-PCR检测MMP-2、MDR1的表达改变。结果:P-AKT、P-gp(MDR1)、MMP-2在MCF-7中为低表达或不表达,MCF-7/ADR’中为高表达。LY294002作用两系后,P-AKT、P-gp(MDR1)、MMP-2在MCF-7/ADR’中的表达明显减低(P<0.05),MCF-7无明显改变。结论:抑制PI3K/AKT信号通路可有效降低MCF-7/ADR’耐药和侵袭转移能力,PI3K/AKT通路是调控乳腺癌多药耐药和侵袭转移的重要信号通路之一。     

冬凌草活性部位逆转SGC7901/ADR细胞多药耐药性的体外研究北大核心CSCD

以SGC7901和SGC7901/ADR为细胞模型,检测了冬凌草活性部位与化疗药物联用后,对SGC7901/ADR耐药性的逆转效应;冬凌草活性部位处理细胞后,检测耐药细胞内阿霉素的蓄积变化、耐药细胞P-糖蛋白(P-gp)的表达水平以及mdr1基因的表达变化。结果显示,冬凌草氯仿部位和乙酸乙酯部位可以有效提高化疗药物阿霉素在SGC7901/ADR细胞内的蓄积,降低P-gp的表达,降低mdr1基因的转录。冬凌草逆转胃癌耐药细胞SGC7901/ADR多药耐药性的活性部位是冬凌草氯仿部位和乙酸乙酯部位,其逆转作用与抑制P-gp的表达相关。     

冬凌草活性部位逆转SGC7901/ADR细胞多药耐药性的体外研究

以SGC7901和SGC7901/ADR为细胞模型,检测了冬凌草活性部位与化疗药物联用后,对SGC7901/ADR耐药性的逆转效应;冬凌草活性部位处理细胞后,检测耐药细胞内阿霉素的蓄积变化、耐药细胞P-糖蛋白(P-gp)的表达水平以及mdr1基因的表达变化。结果显示,冬凌草氯仿部位和乙酸乙酯部位可以有效提高化疗药物阿霉素在SGC7901/ADR细胞内的蓄积,降低P-gp的表达,降低mdr1基因的转录。冬凌草逆转胃癌耐药细胞SGC7901/ADR多药耐药性的活性部位是冬凌草氯仿部位和乙酸乙酯部位,其逆转作用与抑制P-gp的表达相关。     

miR-34c逆转乳腺癌耐药细胞株MCF-7/DOX耐药性的研究

miR-34在肿瘤发生发展中起着至关重要的作用,然而,mi R-34在肿瘤耐药中的作用研究不多。该研究将合成的mi R-34c成熟序列转染乳腺癌阿霉素(doxorubicin,DOX)耐药细胞MCF-7/DOX,探讨mi R-34c体外逆转MCF-7/DOX细胞耐药性作用及其可能的机制。采用Real-time RT-PCR检测mi R-34c在乳腺癌耐药细胞株MCF-7/DOX中的表达,MTS法检测miR-34c对MCF-7/DOX细胞阿霉素耐药性的影响,流式细胞术检测miR-34c对MCF-7/DOX细胞周期和凋亡的影响,Real-time RT-PCR和Western blot法检测多药耐药相关蛋白MDR、MRP以及细胞周期与凋亡相关蛋白Bcl-2、E2F3的表达。结果显示,mi R-34c在乳腺癌MCF-7/DOX耐药细胞中低表达,转染mi R-34c可明显增加耐药细胞对阿霉素的敏感性;流式分析发现,miR-34c可以促进耐药细胞G2期细胞周期阻滞和凋亡;与对照组相比较,miR-34c转染组细胞MDR、MRP蛋白表达无明显变化,而Bcl-2、E2F3 mRNA和蛋白表达均明显下调。研究表明,miR-34c直接靶向抑制Bcl-2和E2F3的表达,诱导细胞周期G2期阻滞和凋亡,进而增强MCF-7/DOX耐药细胞对阿霉素的敏感性。     

穿心莲内酯联合氟康唑抗耐药白假丝酵母菌作用机制研究

目的探讨穿心莲内酯联合氟康唑抗耐药白假丝酵母菌作用及其机制。方法采用微量稀释法检测穿心莲内酯（AG）及联合氟康唑（FLC）对耐药白假丝酵母菌的MIC;采用罗丹明6G（Rh6G）检测AG对耐药白假丝酵母菌CDR外排功能的影响;利用罗丹明123评估AG对耐药白假丝酵母菌MDR外排功能的影响：采用二氢罗丹明检测AG单用及联合FLC对耐药白假丝酵母菌活性氧（ROS）的影响;采用实时荧光定量PCR（qRT—PCR）检测AG联合FLC对耐药白假丝酵母菌外排泵相关基因CDR1、CDR2和MDR1表达的影响。结果AG联合FLC抗耐药白假丝酵母菌呈相加作用;AG对CDR外排功能无影响;AG可抑制MDR外排功能;AG联合FLC能显著提高耐药白假丝酵母菌细胞ROS水平;AG与FLC联合作用于耐药白假丝酵母菌可下调CDR1和MDR1的表达量,上调CDR2的表达量。结论AG联合FLC抗耐药白假丝酵母菌具有相加作用,其机制可能与抑制外排泵及相关基因表达,提高胞内ROS水平有关。     

诱导耐药K562/ADM和转基因耐药K562/MDR细胞株的生物学行为和耐药机制的比较研究

目的探讨转多药耐药基因mdr1的K562/MDR细胞株作为单机制耐药模型的可行性,为进一步研究肿瘤耐药及其逆转奠定基础。方法实验分为3部分:(1)在电子显微镜下观察慢性髓细胞白血病急性红白变敏感细胞系K562,阿霉素(adriamycin,ADM)诱导耐药细胞株K562/ADM和K562/MDR耐药细胞株的生物学行为;同时测定3种细胞系的群体倍增时间;以观察药物诱导和基因转移是否对细胞的生物学行为造成影响。(2)以K562细胞为对照,用MTT法分别测定阿霉素、柔红霉素(daunorubicin,DNR)、长春新碱(vincristine,VCR)对3种细胞的半数致死量(IC50)。(3)多药耐药相关基因与蛋白的检测。免疫细胞化学法观察mdr1基因编码的P-糖蛋白(P-gp)的表达;流式细胞术检测P-gp、bcl-2的表达百分率;生化法测定细胞内谷胱甘肽S-转移酶(GSTs)活性;RT-PCR法检测拓扑异构酶(to-poisomeraseⅡ,topoⅡ)mRNA的表达变化。结果(1)在超微结构上,K562/ADM的细胞器—线粒体出现水肿,K562和K562/MDR未见明显异常;K562的群体倍增时间为19.67±3.10d;K562/MDR为20.40±1.80d;K562/ADM为28.47±1.75d;(2)K562/ADM和K562/MDR细胞对ADM的耐药倍数分别为23.1和1.2倍;对DNR为84.9和14.4倍;对VCR为298.3和10.1倍。(3)与K562比较,K562/ADM细胞的P-gp和Bcl-2蛋白表达率高且topoⅡcDNA片段大小发生变化;K562/MDR仅P-gp表达率高。结论K562/MDR的生物学行为与亲本细胞K562相似,耐药机制单一,可作为单机制耐药模型,对某一耐药基因进行更为深入精确的研究,也可针对该耐药基因准确地筛选相应的逆转剂。     

microRNA-3163靶向乳腺癌耐药蛋白逆转肺癌细胞多药耐药作用

目的:验证microRNA-3163(miR-3163)在肺癌细胞中是否靶向乳腺癌耐药蛋白(BCRP),探索逆转肺癌细胞抗肿瘤药物多药耐药(MDR)的干预策略。方法:检测BCRP在肺癌细胞A549和耐药细胞系A549/ADR中的表达,利用系列浓度梯度的抗肿瘤药物处理细胞,计算其作用的IC50值;在A549/ADR细胞中转染miR-3163的模拟物或抑制剂,用Western印迹检测BCRP的表达水平;在此基础上检测miR-3163对抗肿瘤药物杀伤A549/ADR细胞的影响。结果:与A549细胞相比,A549/ADR细胞具有对抗肿瘤药物的MDR特性,BCRP在A549/ADR细胞中的表达显著上调。转染miR-3163的模拟物能够上调A549/ADR细胞对抗肿瘤药物阿霉素、紫杉醇、吉西他滨和吉非替尼的敏感性,逆转其MDR作用。这些抗肿瘤药物作用的IC50值分别从4.86±0.33、0.41±0.05、3.79±0.26和5.51±0.25μmol/L下调至0.30±0.05、0.07±0.01、0.67±0.10和1.58±0.42μmol/L。特异性实验结果表明,miR-3163的模拟物能够在A549/ADR细胞中下调BCRP的表达水平,转染miR-3163的抑制剂能够阻断miR-3163模拟物的作用。结论:miR-3163有可能通过靶向耐药蛋白BCRP逆转肺癌细胞的MDR作用。     

胃癌SGC7901 表柔比星耐药细胞亚系的建立与耐药机制研究

目的:建立人胃癌SGC7901表柔比星耐药细胞系,探讨其对表柔比星的耐药机制。方法:采用逐步增加表柔比星浓度,间歇作用体外诱导法,建立人胃癌SGC7901表柔比星耐药细胞亚系SGC7901/EPI。用MTT法测定药物敏感性;流式细胞仪检测其药物排除能力和凋亡抵抗能力等生物学指标的改变,western blot检测相关蛋白的表达。结果:经过12个月建成人胃癌SGC7901表柔比星耐药细胞系SGC7901/EPI,其对表柔比星明显耐药,且对其他多种抗癌药具有不同程度的交叉耐药性,阿霉素蓄积潴留实验显示SGC7901/EPI的阿霉素含量明显低于亲本细胞,Western blot显示MRP1的表达上调;SGC7901/EPI凋亡抵抗能力明显上升,Bcl-2表达比亲本细胞增高,而Bax的表达下调。结论:SGC7901/EPI细胞具有多药耐药表型,其可能通过MRP1的上调增加药物排出和上调Bcl-2/Bax的比值促进凋亡抵抗等机制产生耐药。该胃癌多药耐药细胞亚系为进一步研究胃癌耐药机制及逆转方法奠定基础。     

Regulatory mechanism of ZNF139 in multi-drug resistance of gastric cancer cells

Our previous study found increased zinc finger protein 139 (ZNF139) expression in gastric cancer (GC) cells. Purpose of the study is to further clarify the role and mechanism of ZNF139 in multi-drug resistance (MDR) of GC cells. MTT assay, RT-PCR, Western blotting were employed to detect susceptibility of GC cells to chemotherapeutic agents (5-FU, L-OHP) in vitro, and expressions of ZNF139 and MDR associated genes MDR1/P-gp, MRP1, Bcl-2, Bax were also detected. siRNA specific to ZNF139 was transfected into MKN28 cells, then chemosensitivity of GC cells as well as changes of ZNF139 and MDR associated genes were detected. It’s found the inhibition rate of 5-FU, L-OHP to well-differentiated GC tissues and cell line was lower than that in the poorly differentiated tissues and cell line; expressions of ZNF139 and MDR1/P-gp, MRP1 and Bcl-2 in well-differentiated GC tissues and cell line MKN28 were higher, while Bax expression was lower. After ZNF139-siRNA was transfected into MKN28, ZNF139 expression in GC cells was inhibited by 90 %; inhibition rate of 5-FU, L-OHP to tumor cells increased, and expressions of MDR1/P-gp, MRP1 and Bcl-2 were down-regulated, while Bax was up-regulated. ZNF139 was involved in GC MDR by promoting expressions of MDR1/P-gp, MRP1 and Bcl-2 and inhibiting Bax simultaneously.     

A kinetic study of Rhodamine123 pumping by P-glycoprotein

The MDR1 P-glycoprotein (P-gp) actively extrudes a wide variety of structurally diverse cytotoxic compounds out of the cell, is widely expressed in the epithelial cells of kidney, liver and intestine, and in the endothelial cells of brain and placenta, and plays an important role in drug resistance. We measured the accumulation of Rhodamine 123 (Rho123), a substrate of P-gp, into a drug sensitive and a drug resistant strain of the human leukemia cell line K562, as function of Rho123 concentration. With the aid of a mathematical transformation, we used the accumulation of Rho123 into the sensitive cells as a surrogate measure for the internal concentration of the probe in the resistant cells, and were thus able to measure the kinetic parameters of drug efflux pumping by P-gp. Drug pumping was half-saturated at an external Rho123 concentration of 7.2E-06+/-1.1E-06 M, and displayed a co-operative behaviour with a Hill number of 1.94+/-0.32. Verapamil could be shown to inhibit Rho123 efflux uncompetitively.     

A kinetic study of Rhodamine123 pumping by P-glycoprotein

The MDR1 P-glycoprotein (P-gp) actively extrudes a wide variety of structurally diverse cytotoxic compounds out of the cell, is widely expressed in the epithelial cells of kidney, liver and intestine, and in the endothelial cells of brain and placenta, and plays an important role in drug resistance. We measured the accumulation of Rhodamine 123 (Rho123), a substrate of P-gp, into a drug sensitive and a drug resistant strain of the human leukemia cell line K562, as function of Rho123 concentration. With the aid of a mathematical transformation, we used the accumulation of Rho123 into the sensitive cells as a surrogate measure for the internal concentration of the probe in the resistant cells, and were thus able to measure the kinetic parameters of drug efflux pumping by P-gp. Drug pumping was half-saturated at an external Rho123 concentration of 7.2E-06 ± 1.1E-06 M, and displayed a co-operative behaviour with a Hill number of 1.94 ± 0.32. Verapamil could be shown to inhibit Rho123 efflux uncompetitively.     

Clitocine Reversal of P-Glycoprotein Associated Multi-Drug Resistance through Down-Regulation of Transcription Factor NF-κB in R-HepG2 Cell Line

Multidrug resistance(MDR)is one of the major reasons for failure in cancer chemotherapy and its suppression may increase the efficacy of therapy. The human multidrug resistance 1 (MDR1) gene encodes the plasma membrane P-glycoprotein (P-gp) that pumps various anti-cancer agents out of the cancer cell. R-HepG2 and MES-SA/Dx5 cells are doxorubicin induced P-gp over-expressed MDR sublines of human hepatocellular carcinoma HepG2 cells and human uterine carcinoma MES-SA cells respectively. Herein, we observed that clitocine, a natural compound extracted from Leucopaxillus giganteus, presented similar cytotoxicity in multidrug resistant cell lines compared with their parental cell lines and significantly suppressed the expression of P-gp in R-HepG2 and MES-SA/Dx5 cells. Further study showed that the clitocine increased the sensitivity and intracellular accumulation of doxorubicin in R-HepG2 cells accompanying down-regulated MDR1 mRNA level and promoter activity, indicating the reversal effect of MDR by clitocine. A 5'-serial truncation analysis of the MDR1 promoter defined a region from position -450 to -193 to be critical for clitocine suppression of MDR1. Mutation of a consensus NF-κB binding site in the defined region and overexpression of NF-κB p65 could offset the suppression effect of clitocine on MDR1 promoter. By immunohistochemistry, clitocine was confirmed to suppress the protein levels of both P-gp and NF-κB p65 in R-HepG2 cells and tumors. Clitocine also inhibited the expression of NF-κB p65 in MES-SA/Dx5. More importantly, clitocine could suppress the NF-κB activation even in presence of doxorubicin. Taken together; our results suggested that clitocine could reverse P-gp associated MDR via down-regulation of NF-κB.     

Chloride channel-3 mediates multidrug resistance of cancer by upregulating P-glycoprotein expression

Chloride channel-3 (ClC-3), a member of the ClC family of voltage-gated Cl⁻ channels, is involved in the resistance of tumor cells to chemotherapeutic drugs. Here, we report a new mechanism for ClC-3 in mediating multidrug resistance (MDR). ClC-3 was highly expressed in the P-glycoprotein (P-gp)-dependent human lung adenocarcinoma cell line (A549)/paclitaxel (PTX) and the human breast carcinoma cell line (MCF-7)/doxorubicin (DOX) resistant cells. Changes in the ClC-3 expression resulted in the development of drug resistance in formerly drug-sensitive A549 or MCF-7 cells, and drug sensitivity in formerly drug-resistant A549/Taxol and MCF-7/DOX cells. Double transgenic MMTV-PyMT/CLCN3 mice with spontaneous mammary cancer and ClC-3 overexpression demonstrated drug resistance to PTX and DOX. ClC-3 expression upregulated the expression of MDR1 messenger RNA and P-gp by activating the nuclear factor-κB (NF-κB)-signaling pathway. These data suggest that ClC-3 expression in cancer cells induces MDR by upregulating NF-κB-signaling-dependent P-gp expression involving another new mechanism for ClC-3 in the development of drug resistance of cancers.     

Carotenoids reverse multidrug resistance in cancer cells by interfering with ABC-transporters

Proteins of the ATP-binding cassette superfamily, mainly P-glycoprotein (P-gp; MDR1), play an important role in the development of multidrug resistance (MDR) in cancer cells and thus in the potential failure of chemotherapy. A selection of carotenoids (β-carotene, crocin, retinoic acid, canthaxanthin, and fucoxanthin) was investigated whether they are substrates of P-gp, and if they can reverse MDR in resistant Caco-2 and CEM/ADR5000 cells as compared to the sensitive parent cell line CCRF-CEM. The activity of ABC transporter was determined in resistant and sensitive cells by spectrofluorometry and flow cytometry using the substrates doxorubicin, rhodamine 123, and calcein as fluorescent probes. The carotenoids increased accumulation of these P-gp substrates in a dose-dependent manner indicating that they themselves also function as substrates. Fucoxanthin and canthaxanthin (50-100μM) produced a 3-5-fold higher retention of the fluorescent probes than the known competitive inhibitor verapamil. Carotenoids showed a low cytotoxicity in cells with MDR with IC(50) values between 100 and 200μM. The combination of carotenoids with eight structurally different cytotoxic agents synergistically enhanced their cytotoxicity in Caco-2 cells, probably by inhibiting the function of the ABC transporters. For example, fucoxanthin synergistically enhanced the cytotoxicity of 5-FU 53.37-fold, of vinblastine 51.01-fold, and of etoposide 12.47-fold. RT-PCR was applied to evaluate the mRNA levels of P-gp in Caco-2 cells after treatment with carotenoids. Fucoxanthin and canthaxanthin significantly decreased P-gp levels to 12% and 24%, respectively as compared to untreated control levels (p<0.001). This study implies that carotenoids may be utilised as chemosensitisers, especially as adjuvants in chemotherapy.